DNA damaging agents of diverse origins continuously challenge the genome of living organisms and impact its normal function. Eukaryotes counteract by deploying an effective DNA damage response (DDR) by promptly activating cell cycle checkpoints, initiating DNA repair and reassembling the intact chromatin. This intricate phenomenon depends on sequential recruitment and timely clearance of a number of cross- talking factors. During the previous project period, we have identified ATM checkpoint kinase (a master cell cycle checkpoint controller), and CRL4DDB2 ubiquitin ligase complex (a helix-distorting DNA lesion sensor), as the two key regulators of DNA repair-dependent chromatin assembly. The underlying premise of this renewal proposal is that DNA damage activates parallel events that impinge on (i) arresting cells to allow access to DNA damage and repair machinery and (ii) restoration of epigenetically intact chromatin and resumption of normal cell cycling. Accordingly the studies are designed to focus on (1) uncovering the molecular underpinning of ATM-directed H3K56ac-driven repair-dependent chromatin assembly, and (2) elucidate the function and mechanism of CRL4DDB2 ubiquitin ligase complex in integrated events of histone acetylation, ubiquitination and deposition in chromatin. The results would prove the related hypothesis that (a) ATM kinase facilitates repair-dependent chromatin assembly via a novel phosphorylation signaling cascade, and (b) CRL4DDB2 ubiquitin ligase directs the H3K56 acetylation and histone deposition during chromatin restoration. The proposed work will utilize a series of relevant technologies to address following inter-linked specific objectives: (1) to establish the role of ATM-mediated DNA damage signaling in regulating repair-dependent chromatin assembly, (2) to elucidate the function of CRL4DDB2 Ub-ligase in H3K56 acetylation, and (3) to establish the mechanism of CRL4DDB2 Ub-ligase-mediated regulation of post- repair histone deposition in chromatin restoration. Variety of human cell lines, normal or specifically lacking individual protein factors, either constitutively or by siRNA/shRNA mediated gene silencing, will be utilized at select stages of cell cycle to analyze the effects on checkpoint protein factors and reveal their functional interactions through FACS analysis, ChIP, co-immunoprecipitation and/or by co-localization assays. A battery of histone modifications will be evaluated in specifically compromised cells to reveal the alterations that regulate DNA repair and cell cycle progression. Lastly, purified recombinant histones, acetyltransferases, and CRL4DDB2 will be tested in vitro to delineate their specific biochemical roles in vivo. These systematic mechanistic studies of DDR would not only provide the knowledge relevant to cancer etiology, but also new amenable tools, targets and strategies for managing human health risk.
The project goal is to address the ultimate concern of how humans effectively respond to deleterious environmental exposures. The systematic studies are designed to understand the key events of DNA damage response which is promptly launched by human cells to activate cell cycle checkpoints for allowing the DNA repair and restoration of chromatin and its normal function. The insights developed through the proposed studies will help develop new amenable tools, targets and strategies for managing human health risk.
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